King's College London

Research portal

SARS-CoV-2 Spike S1 glycoprotein is a TLR4 agonist, upregulates ACE2 expression and induces pro-inflammatory M1 macrophage polarisation: Preprint, available at: doi: https://doi.org/10.1101/2021.08.11.455921;

Research output: Contribution to journalArticle

Original languageEnglish
Article numberdoi: https://doi.org/10.1101/2021.08.11.455921;
Pages (from-to)1
Number of pages30
JournalbioRxiv
DOIs
Published11 Aug 2021

Bibliographical note

Preprint in BioRxiv.

King's Authors

Abstract

Background and aims: TLR4 is an important innate immune receptor that recognizes bacterial LPS, viral proteins and other pathogen associated molecular patterns (PAMPs). It is expressed on tissue-resident and
immune cells. We previously proposed a model whereby SARS-CoV-2 activation of TLR4 via its spike glycoprotein S1 domain increases ACE2 expression, viral loads and hyperinflammation with COVID-19 disease [1]. Here we test this hypothesis in vitro and demonstrate that the SARS-CoV-2 spike S1 domain is
a TLR4 agonist in rat and human cells and induces a pro-inflammatory M1 macrophage phenotype in human THP-1 monocyte-derived macrophages. Methods: Adult rat cardiac tissue resident macrophage-derived fibrocytes (rcTMFs) were treated with either bacterial LPS or recombinant SARS-CoV-2 spike S1 glycoprotein. The expression of ACE2 and other inflammatory and fibrosis markers were assessed by immunoblotting. S1/TLR4 co-localisation/binding was assessed by immunocytochemistry and proximity
ligation assays on rcTMFs and human HEK-293 HA-TLR4-expressing cells. THP-1 monocytes were differentiated into M1 or M2 macrophages with LPS/IFN, S1/IFN or IL-4 and RNA was extracted for RT-qPCR of M1/M2 markers and ACE2. Results: TLR4 activation by spike S1 or LPS resulted in the upregulation
of ACE2 in rcTMFs as shown by immunoblotting. Likewise, spike S1 caused TLR4-mediated induction of the inflammatory/wound healing marker COX-2 and concomitant downregulation of the fibrosis markers CTGF and Col3a1, similar to LPS. The specific TLR4 TIR domain signalling inhibitor CLI-095 (Resatorvid®), blocked the effects of spike S1 and LPS, confirming that spike S1 is a TLR4 agonist and viral PAMP (VAMP). ACE2 expression was also inhibited by the dynamin inhibitor Dynasore®, suggesting ACE2 expression is mediated
by the alternative endosomal/β-interferon pathway. Confocal immunofluorescence microscopy confirmed 1:1 stoichiometric spike S1 co-localisation with TLR4 in rat and human cells. Furthermore, proximity
ligation assays confirmed spike S1 and TLR4 binding in human and rat cells. Spike S1/IFN-γ treatment of THP-1-derived macrophages induced pro-inflammatory M1 polarisation as shown by an increase in IL-1β
and IL-6 mRNA. Conclusions: These results confirm that TLR4 is activated by the SARS-CoV-2 spike protein S1 domain and therefore TLR4 may be a receptor/accessory factor for the virus. By binding to and activating TLR4, spike S1 caused upregulation of ACE2, which may facilitate viral entry into cells. In
addition, pro-inflammatory M1 macrophage polarisation via TLR4 activation, links TLR4 activation by spike S1 to inflammation. The clinical trial testing of CLI-095 (Resatorvid®) and other TLR4 antagonists in severe COVID-19, to reduce both viral entry into cells and hyperinflammation, is warranted. Our findings likely represent an important development in COVID-19 pathophysiology and treatment, particularly regarding cardiac complications and the role of macrophages.

View graph of relations

© 2020 King's College London | Strand | London WC2R 2LS | England | United Kingdom | Tel +44 (0)20 7836 5454